International Journal of

Molecular Sciences

Review

Insulin Signal Transduction Perturbations in Insulin Resistance

Mariyam Khalid 1, Juma Alkaabi 2 , Moien A. B. Khan 3 and Abdu Adem 1,4,*

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Citation: Khalid, M.; Alkaabi, J.;

Khan, M.A.B.; Adem, A. Insulin

Signal Transduction Perturbations in

Insulin Resistance. Int. J. Mol. Sci.

2021, 22, 8590. https://doi.org/

10.3390/ijms22168590

Academic Editor: Gerhard Kostner

Received: 31 May 2021

Accepted: 28 July 2021

Published: 10 August 2021

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1 Departments of Pharmacology, College of Medicine and Health Sciences, United Arab Emirates University,Al Ain P.O. Box 17666, United Arab Emirates; 201690406@uaeu.ac.ae

2 Departments of Internal Medicine, College of Medicine and Health Sciences,United Arab Emirates University, Al Ain P.O. Box 17666, United Arab Emirates; j.kaabi@uaeu.ac.ae

3 Departments of Family Medicine, College of Medicine and Health Sciences, United Arab Emirates University,Al Ain P.O. Box 17666, United Arab Emirates; moien.khan@uaeu.ac.ae

4 Department of Pharmacology, College of Medicine and Health Sciences, Khalifa University,Abu Dhabi P.O. Box 127788, United Arab Emirates

* Correspondence: abdu.adem@ku.ac.ae

Abstract: Type 2 diabetes mellitus is a widespread medical condition, characterized by high bloodglucose and inadequate insulin action, which leads to insulin resistance. Insulin resistance in insulin-responsive tissues precedes the onset of pancreatic β-cell dysfunction. Multiple molecular andpathophysiological mechanisms are involved in insulin resistance. Insulin resistance is a consequenceof a complex combination of metabolic disorders, lipotoxicity, glucotoxicity, and inflammation. Thereis ample evidence linking different mechanistic approaches as the cause of insulin resistance, butno central mechanism is yet described as an underlying reason behind this condition. This reviewcombines and interlinks the defects in the insulin signal transduction pathway of the insulin resistancestate with special emphasis on the AGE-RAGE-NF-κB axis. Here, we describe important factors thatplay a crucial role in the pathogenesis of insulin resistance to provide directionality for the events. Theinterplay of inflammation and oxidative stress that leads to β-cell decline through the IAPP-RAGEinduced β-cell toxicity is also addressed. Overall, by generating a comprehensive overview of theplethora of mechanisms involved in insulin resistance, we focus on the establishment of unifyingmechanisms to provide new insights for the future interventions of type 2 diabetes mellitus.

Keywords: insulin resistance; hyperglycemia; type 2 diabetes mellitus; insulin signaling; pancreaticbeta cells

1. Introduction

Type 2 diabetes mellitus (T2DM) is a serious medical challenge of the 21st century.Persistently elevated glucose concentrations above the physiological range result in themanifestation of diabetes. The peptide hormone insulin released from pancreatic β-cellsmaintains normal blood glucose levels by regulating whole-body carbohydrate, lipid,and protein metabolism [1]. Insulin performs its function through signal transduction ininsulin-responsive tissues, specifically the liver, skeletal muscles and adipose tissues asthese tissues play a distinct role in the regulation of metabolic homeostasis [2,3].

Insulin resistance is the condition in which a cell, tissue, or body of an organismcannot adequately respond to normal levels of insulin. This in turn hinders insulin’s func-tion of maintaining glucose and lipid homeostasis [4]. The inability of insulin to providenormoglycemia leads to compensatory hyperinsulinemia, which further enhances insulinsecretion from β-cells. Insulin secretion for a prolonged period exhausts pancreatic β-cellsand leads to their apoptosis [5,6]. Insulin resistance also promotes gluconeogenesis inthe liver, disrupts glucose uptake in muscles, and induces lipolysis in adipose tissues.Insulin resistance may be developed through both genetic and acquired factors [7]. Thecommon genetic defects include mutations and polymorphism of insulin receptors, glucosetransporters, and signaling proteins involved in insulin signal transduction. The acquired

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causes of insulin resistance include obesity, physical inactivity, advanced glycation endproducts (AGE), excess free fatty acids (FFAs), psychological stress, smoking, alcoholintake, or certain medications [8–10]. All these factors are linked to constant low-gradeinflammatory conditions [11,12]. The sustained elevation of interleukin-1 beta (IL-1β),interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or FFAs impairs the balance of secre-tion between inflammatory and anti-inflammatory cytokines. This causes accumulationof unfolded protein, leading to activation of unfolded protein response (UPR), inductionof endoplasmic reticulum stress (ER stress), and oxidative stress, which further impairsinsulin sensitivity [13–15].

In this review, we mainly focus on the perturbations of downstream critical nodesof the insulin signaling mechanisms in an insulin-resistant state. Insulin resistance is aconsequence of a complex combination of metabolic disorders, lipotoxicity, glucotoxicity,and inflammation. At the insulin receptor site, the insulin signal transduction includesvarious upstream and downstream modulator proteins. Alterations in the regulation ofinsulin signaling mechanisms are the final manifestation of insulin resistance [16–18]. Here,we attempt to unravel the detailed circuitry of the PI3K-Akt signaling network concerningpathological features elicited by its dysregulation to guide further mechanistic investiga-tions. Special attention is given to mediators that link cellular mechanisms associated withthe pathophysiology of insulin resistance. This review aims to provide a better under-standing and insight for the future interventions of diabetes mellitus. Treatments shouldnot only target the insulin signaling pathway perturbations but should also aim at theAGE-RAGE-NF-κB axis, lipotoxicity, inflammation, and oxidative stress. In this review,we hope that a more sophisticated understanding of the mechanistic link between insulinresistance and its associated key pathophysiological processes presents the molecular basisto facilitate the development of novel therapies for type 2 diabetes mellitus.

2. Insulin Receptor and Insulin Signaling Pathway

Insulin receptor (IR) is a tyrosine kinase receptor that undergoes autophosphorylationupon insulin binding. Structurally, IR is a transmembrane heterotetrameric glycoproteinthat has two α and two β subunits linked with a disulfide bond [19]. The IR is virtuallyexpressed on the surface of all tissues. Major targets are insulin-responsive organs such asthe liver, skeletal muscle, and adipose tissue [20]. The IR has an extracellular portion thatis associated with insulin binding and an intracellular portion that is associated with itstyrosine kinase activity. As the insulin binds to the extracellular portion of the α subunit,oligomerization of the receptor and autophosphorylation of its tyrosine residues amplifiesthe kinase activity of the IR [21,22]. Along with the phosphorylation of IR, insulin receptorsubstrate (IRS), Casitas B-lineage lymphoma (Cbl), or Cbl associated proteins, which are thescaffolding proteins, also get phosphorylated and bind to intracellular receptor sites [23].The IRS is the connecting molecule that, after phosphorylation, recruits signaling moleculesto generate the insulin signal response and promote the activation of different down-stream signaling pathways as the phosphoinositide-3-kinase (PI3K) or the CAP/Cbl/TC10pathway. PI3K is a class 1 phosphoinositide 3-kinase and once activated causes the gen-eration of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which has multiple effectson insulin-dependent glucose metabolism [17] (Carnagarin et al., 2015). PIP3 docks thepleckstrin homology (PH) domain containing protein kinases, which, in turn, activatesphosphoinositide-dependent kinase 1 (PDK1) and protein kinase B or Akt kinase [17,24].Upon activation, Akt migrates to the plasma membrane and phosphorylates Akt sub-strate of 160 kDa (AS160) [25,26]. As a consequence, glucose transporter protein (GLUT)is trafficked from intracellular storage vesicles to the plasma membrane [27] (Figure 1).Insulin-mediated glucose uptake and storage under anabolic conditions in insulin-sensitivetissues are facilitated by the recruitment of GLUT to the cell surface, which is governed bydistinct signaling pathways and is crucial for insulin sensitivity [25,28].

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Figure 1. Various pathways involved in dysregulation of insulin signaling. Upon insulin bind-ing, insulin receptor gets autophosphorylated. This recruits different substrate adaptors for thesignal transduction. The tyrosine phosphorylated IRS1 recruits PI3K, which phosphorylates theserine/threonine residue of protein kinase B (Akt). Akt regulates the translocation of glucose trans-porter GLUT4 to the cell surface through phosphorylation of the GTPase-activating protein (AS160).Akt promotes glycogen synthesis through inhibition of GSK3 activity and induces protein synthesisvia activation of mTOR and downstream elements. Akt phosphorylates and directly inhibits FoxOtranscription factors, which inhibits autophagy. The hyperglycemia-induced production of AGEand binding with their receptor RAGE impairs insulin signal transduction by triggering a range ofsignaling pathways, including JNK, NF-κB, and activation of PKC. The sustained accumulation ofAGE depletes the expression of anti-AGE cell surface receptor AGER1, which is responsible for in-hibiting the deleterious effects of AGE by competitively interfering with its binding to RAGE. AGER1along with Sirtuin1 promotes AMPK phosphorylation and activation, which induces GLUT4 geneexpression through activation of MEF, GEF transcription factors. The AGE-RAGE induced activationof PKC, NF-κB mediated inflammation, and oxidative stress promotes the serine phosphorylationof IRS, inhibits its action, and induces insulin resistance. AGE: Advanced glycation end products;AGER1: AGE receptor 1 encoded by DDOST gene; AMPK: AMP-activated protein kinase; AS160: Aktsubstrate of 160 kDa; FoxO: Forkhead family of transcription factors; GLUT4: Glucose transporterprotein 4; GEF: GLUT4 enhancer factor; GSK3: Glycogen synthase kinase 3; IKK: IκB kinase; IRS:Insulin receptor substrate1; MEF: Myocyte enhancer factor; mTOR: Mammalian target of rapamycinNF-κB: Nuclear factor κB; PDK1: Phosphoinositide-dependent kinase 1; PI3K: Phosphoinositide3-kinase; PIP3: Phosphatidylinositol (3,4,5)-trisphosphate; PKC: Protein kinase C; RAGE: Receptorfor advanced glycosylation end products; S6K: Ribosomal protein S6 kinase; SREBP: Sterol regulatoryelement-binding proteins; JNK: c-Jun N-terminal kinase.

3. Mechanisms Involved in Insulin Resistance3.1. β-Cell Function and Mass

Insulin secretion and insulin sensitivity are regulated by pancreatic β-cells in a verydefinite manner to maintain homeostatic concentrations of plasma glucose in healthyindividuals. During normal physiological conditions, there is a positive feedback loopbetween the β-cells and insulin-sensitive tissues with enhanced insulin supply by β-cells in response to demand by the liver, skeletal muscle, and adipose tissue [5,29]. Themagnitude of insulin response from β-cells depends on the sensitivity of insulin-responsivetissues [30,31]. The intimate, highly complex link between insulin resistance and β-celldysfunction has important roles in triggering the pathogenesis of type 2 diabetes mellitus.The exhaustion of β-cells to maintain euglycemia and compensate for insulin demand inthe insulin-resistant state is critical to the pathogenesis of the condition [29].

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Both in rodents and humans, FFAs are crucial for glucose-stimulated insulin secretion(GSIS) from pancreatic β-cells through to the activation of G-protein coupled receptorsspecifically GPR40 [32]. During the insulin-resistant phase, which is characterized bylipotoxicity and elevated FFAs, overstimulation of glucose-mediated insulin secretion inβ-cells results in increased β-cell signaling and oxidative stress, which leads to metabolicexhaustion of beta cells [33,34]. Glucose along with elevated FFAs leads to an increase inβ-cell mass by increasing IRS2 expression of β-cells, which is associated with neogenesis,proliferation, and survival of pancreatic β-cells as a compensatory mechanism for insulinresistance. However, the resulting hyperinsulinemia leads to β-cell dysfunction [35]. Theratio of proinsulin and insulin split products relative to total immunoreactive insulin isincreased in type 2 diabetes mellitus patients reflecting their β-cell dysfunction comparedwith healthy individuals [36]. The deposition of islet amyloid polypeptide (IAPP) or amylinin pancreatic islets is the other pathological mechanism involved in β-cell dysfunction.IAPP deposition causes a progressive decline in pancreatic islet cell mass, predominantly β-cells. Recently it was identified that the increase in expression of the receptor for advancedglycation end products (RAGE) contributes to IAPP induced β-cell proteotoxicity, asRAGE by binding to IAPP toxic intermediates triggers oxidative stress, inflammation, andapoptosis that are known to be upregulated in IAPP induced β-cell pathogenesis [37]. Theaccumulation and binding of ligands, i.e., AGE and IAPP toxic intermediates, upregulateRAGE expression and transduce RAGE-mediated intracellular signaling, leading to isletinflammation and β-cell apoptosis [33,37,38].

Increased cellular glucose metabolism, accumulation of saturated long chain fatty acidsignaling, defective proinsulin processing, abnormal insulin secretion, the decline in β-cellmass, and islet amyloid deposition are some of the key factors in an insulin-resistant statethat contribute to β-cell demise and lead to disease progression [39].

3.2. Insulin Receptor Substrate

The IRS is a critical mediator of insulin action and is a major site for both positiveand negative regulation of the insulin signaling pathway. The IRS family has two majorproteins IRS1 and IRS2, which function as docking proteins between the insulin receptorand intracellular signaling cascade [40,41]. IRS1 and IRS2 are significantly divergent in theirabundance and tissue-specific function. Significantly lower expression of IRS1 has beenobserved in morbidly obese and insulin-resistant subjects [42]. IRS1-dependent insulinsignal transduction is predominant over IRS2 in skeletal muscle, which has a significant roleto maintain whole-body metabolism [43]. Insulin-mediated IRS tyrosine phosphorylation isthe key intermediate in insulin signal transduction and serves as the major target for insulinresistance inducers [41]. Under normal physiological conditions, the insulin signaling alsoinduces the IRS1 serine phosphorylation at specific sites as a negative feedback controlmechanism through the activation of several kinases. Regulation of insulin/IGF-1 signalingby IRS involves phosphorylation at serine/threonine residues, dephosphorylation by thephosphatase, and degradation through the ubiquitin-proteasome [20,44].

The gene encoding IRS1 is highly polymorphic, and some notable single nucleotidepolymorphisms (SNPs) are reported in insulin-resistant subjects [43,45]. These mutationsreduce the extent of phosphorylation of IRS1 and the insulin stimulated PI3K activity,leading to impaired insulin action. In insulin resistance, IRS1 malfunctions more oftenthan IRS2 [40,41]. IRS1 and IRS2 share a high degree of structural homology, but theydiffer in their activation sites and cellular localization [40]. IRS1 has an N-terminal PHdomain, which is flanked by the phosphotyrosine binding (PTB) domain, responsible for itsinteraction with IR. The tail of IRS1 contains distinct tyrosine and serine phosphorylationsites, which serve as the docking locus for SH2 homology signal transducers [20,46].The “diabetogenic” factors, specifically FFAs, inflammatory cytokines, reactive oxygenspecies, and hyperinsulinemia, strikingly increase various serine kinases, which includeIκB kinase (IKK), c-Jun N-terminal kinase (JNK), specific isoforms of protein kinase C(PKC), double-stranded RNA-dependent protein kinase (PKR), and Rho-associated coiled-

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coil containing protein kinase (ROCK) [47]. These stress kinases not only hamper theIRS1 function but promote insulin resistance through upregulation of the expression ofgenes involved in the activation of inflammation and nuclear factor kappa B (NF-κB),thus further intensifying the problem. Phosphorylation of IRS1 at its serine residues,specifically on Ser307, Ser612, and Ser632, serves as a core element in the development ofinsulin resistance [41]. This phosphorylation cascade is a complex regulatory process thatinhibits IRS1 tyrosine phosphorylation resulting in dissociation of the IR:IRS1 complex andpromotes IRS1 de-localization and degradation [41,48]. In the light of above-mentioneddata, the strategic intervention that can upregulate the expression of IRS1 and has functionalsignificance can alleviate insulin resistance-induced skeletal muscle pathological damage.Moreover, detection of IRS1 novel variants that substantially contribute to the developmentof insulin resistance needs to be assessed.

3.3. Phosphatidylinositol 3-Kinase

Following the tyrosine-phosphorylation of IRS, Phosphatidylinositol 3-kinase (PI3K)is an important effector molecule. PI3K is a heterodimer having serine kinase activity,which enables it to interact with other signaling proteins [49]. PI3K is a key regulatorynode in translating extracellular signals activated by insulin and growth factors intointracellular actions [50]. The class 1A PI3K comprises two subunits, 85-kDa regulatorysubunit and a 110-kDa catalytic subunit. The regulatory subunit is responsible for thenegative regulation of PI3K and has three isoforms: p85α, p85β, and p55γ [17]. The p85subunit interacts with tyrosine-phosphorylated IRS through its two SH2 domains, andthe p110 subunit hydrolyzes the membrane-bound phosphatidylinositol 4,5-bisphosphate(PIP2). The conversion of PIP2 to PIP3 is crucial for the recruitment of PDK1, which isresponsible for phosphorylation of Akt that stimulates insulin signal transduction [51].

The balanced expression level of the p85 monomer and p85-p110 heterodimer is acritical feature of PI3K activation [52,53]. Insulin resistance is mediated when the expressionof p85α monomer is enhanced as it competes with p85-p110 heterodimer and sequestersIRS1 and recruits inhibitory proteins responsible for PIP3 degradation. The p85α is encodedby PIK3R1. Mutations in PI3KR1 are associated with reduced PI3K signaling and anescalated risk of type 2 diabetes mellitus [54,55]. The combined outcome of enhanced p85subunit expression and IRS serine phosphorylation is adequate to develop clinical insulinresistance [17,50,56].

Further investigation of the mechanisms that hamper binding of the p85-p110 het-erodimer to the insulin receptor, block the activation of catalytic subunit p110, and causemutations in the regulatory subunit of PI3K may serve as a guide for finding possiblemolecular targets for the prevention of impaired insulin signaling.

3.4. Protein Kinase B/Akt

The serine/threonine protein kinase Akt (also known as protein kinase B or PKB) is amember of the AGC protein kinases family. Akt consists of three homologous isoforms,i.e., Akt1, Akt2, and Akt3. The three isoforms share structural similarities but exhibitdiverse target specific roles [57]. Among all the isoforms of Akt, Akt2 is the most importantone in insulin-mediated glucose uptake and lipid metabolism. PDK1 initiates the Aktactivation by phosphorylation of Akt at its threonine residue; subsequently, mTORC2completes the Akt activation process by phosphorylation of Akt at its serine residue [25,58].Phosphorylation at Thr308 and Ser473 is required for the maximal activation of Akt. Aktdirectly phosphorylates AS160, limiting the activity of the Rab-GTPase activating protein,leading to GLUT4 translocation and glucose uptake. Akt-mediated activation of mTORC1through inhibition of the tumor suppressor complex (TSC1/TSC2) regulates several pro-teins involved in protein synthesis, including ribosomal protein p70, ribosomal S6 kinase1 (S6K1) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), bythe acceleration of mRNA translation [17]. Akt exerts an inhibitory effect on glycogensynthase kinase 3 (GSK3) by phosphorylation resulting in glycogen synthase activation.

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Akt through mTORC1 activation suppresses lipolysis and promotes lipid biosynthesis byregulating the sterol regulatory element-binding protein (SREBP) substrate. Akt interactsand phosphorylates forkhead box transcription factor (FoxO) proteins, which are involvedin the regulation of lipogenic and gluconeogenic gene expression, particularly FoxO1 andFoxO3. Akt attenuates the transcriptional activity of FoxO1 through its translocation fromthe nucleus, thus reducing glucose levels [59–61] (Figure 1).

Akt plays a crucial role in cell survival and growth through its action on insulin-stimulated glucose uptake, glycogen, and protein synthesis. The depletion of p-Akt expres-sion is physiologically regulated for metabolic homeostasis either through the negativefeedback by S6K1 or by the intracellular signaling molecules known as inositol phos-phates [62,63]. The inositol phosphate, specifically inositol pyrophosphate IP7 generated byinositol-hexakisphosphate (IP6) kinase 1 (IP6K1), directly by its binding to the PH domainof Akt competes for PIP3 binding to Akt [64]. Studies have shown that the IP6K1-deletedmice phenotype shows enhanced Akt activity, sustained insulin sensitivity, and resistanceto obesity as compared to its wild type [64,65]. Deregulated inositol metabolism leadsto Akt inhibition and is associated with hyperglycemia and insulin resistance [66]. It isevident from both human and animal research that knockdown or depletion of either Akt1or Akt2 isoform causes glucose intolerance, insulin resistance, and diabetes-like symptoms.Any defect in the PI3K/Akt/AS160 signaling will eventually reduce the glucose uptake ininsulin-sensitive tissues and lead to insulin resistance [21,67].

Since the discovery of Akt in the early 1990s, a remarkable understanding of Akt’srole in the regulation, function, and manipulation of insulin signal transduction has beenmade. The cross-talk of Akt with other major cellular signaling networks has indicated Aktas an attractive candidate for further mechanistic exploration. In this regard, regulatoryphosphorylation events on Akt need to be explored as most studies have been centered ontwo major regulatory sites Thr308 and Ser473 on Akt.

3.5. GLUT4

Glucose transporter proteins (GLUTs) are fundamental membrane proteins that are re-sponsible for glucose transport across the plasma membrane into cells. There are 13 glucosetransporter proteins expressed in distinct tissues having different kinetics and respectivesubstrate specificities. GLUT4 is the predominant insulin-responsive glucose transporterresponsible for the regulation of glucose disposal to maintain whole-body glucose home-ostasis [68,69]. Upon insulin signal transduction, GLUT4 translocates from intracellularstorage vesicles to the cell membrane to enhance glucose uptake. The expression lev-els of GLUT4 mRNA and protein affect insulin sensitivity and can modify whole-bodymetabolism [70]. It has been shown that the condition of insulin deficiency or resistancecan result in depletion of GLUT4 expression in skeletal muscle due to transcriptionalrepression as in T1DM and T2DM. NF-κB, characterized as the mediator of TNF-α, is an im-portant inhibitor of GLUT4 transcription. Exercise and muscle differentiation are the potentphysiological stimulus for upregulation of GLUT4 expression and translocation [71–73].

Different research studies conducted both in tissue culture cell lines and in trans-genic mice have demonstrated that the regulation of GLUT4 gene expression is under thedynamic control of specific transcription factors, the GLUT4 enhancer factor (GEF), andmyocyte enhancer factor 2 (MEF2) [74]. The binding of heterodimer MEF2A-MEF2D to theconsensus sequence in the GLUT4 promoter region upregulates GLUT4 expression and anydeletion or truncation of that specific consensus sequence leads to repression in GLUT4mRNA expression [71,75]. The transcriptional activation of MEF2 is regulated by diversemechanisms, such as calcium levels in the muscle, dephosphorylation by calcineurin, andAMPK activation, which increases PPARβ via MEF2A [76,77].

As GLUT4 expression is much decreased in insulin-resistant state and T2DM, potentstimulators of GEF and MEF2 can be used as an alternative treatment to enhance GLUT4 ex-pression for improved insulin sensitivity as experimentally demonstrated in some researchstudies through the use of GLUT4 overexpression in murine skeletal muscle [70,77,78].

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Studies have shown that moderate overexpression of GLUT4 in both in vitro and in vivomodels positively manipulates glucose uptake and alleviates lipotoxicity [79,80].

Despite intensive efforts to understand the GLUT4 pathway, certain gaps in theregulation of GLUT4 trafficking and signaling components involved in the regulation ofGLUT4 translocation remain elusive. Future investigation to better understand moleculardetails of GLUT4 regulation may lead closer to the advent of selective pharmacologicalintervention.

3.6. Mammalian Target of Rapamycin/mTOR

The mammalian target of rapamycin (mTOR) regulates cellular growth and metabolismthrough its serine/threonine kinase activity. mTOR forms two functionally distinct com-plexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both these multi-protein complexes are activated by growth factors and insulin [81,82].

mTORC1 consists of five components. Insulin-IRS mediated activation of Akt resultsin indirect activation of mTORC1, which, in turn, phosphorylates two key components; the4E-BP1 and the S6K1. Upon phosphorylation, 4EBP1 dissociation from eIF4E allows eIF4Eto promote 5′ cap-dependent mRNA translation leading to protein synthesis. Activationof S6K1 by mTORC1 not only leads to an increase in mRNA biogenesis and translationinitiation but is also involved in negative feedback regulation of insulin signaling throughserine/threonine phosphorylation of IRS, which ultimately leads to IRS depletion [83–85](Figure 1). Chronic activation of mTORC1, as in the case of overnutrition or obesity, leadsto insulin resistance through a negative feedback loop through IRS degradation. One studyconducted in mice deficient in S6K1 demonstrated that mice were protected against insulinresistance under high-fat diet (HFD) conditions, which was apparent in the loss of negativeregulation from S6K1 through its serine phosphorylation of IRS1 [86,87]. In line with thenegative feedback role of mTORC1/S6K1, it has a crucial role in the growth and survival ofβ-cell and insulin secretion. β-cell mass is reduced in chronic administration of rapamycin(inhibitor of mTOR) in rats, and S6K1 knockout mice tend to have small-sized pancreatic βcells [84].

The mTORC2 complex consists of seven protein components, most of which are similarto mTORC1. In comparison to mTORC1, little is known about the mTORC2 pathway.mTORC2 plays a positive role in cell survival and cytoskeleton organization. As mTORC2activation is dependent on the insulin-PI3K transduction pathway, that is why it is affectedby the mTORC1/S6K1 negative feedback loop through phosphorylation of mammalianSAPK interacting protein 1 (mSin1), a key component of mTORC2 [88]. Partially activeAkt promotes the activation of mTORC2, which, in turn, phosphorylates several kinases,including Akt and protein kinase C α (PKCα). mTORC2 regulates glucose homeostasisthrough Akt, which is involved in GLUT4 translocation and deactivation of GSK-3, thusdecreasing the rate of phosphorylation of glycogen synthase. This leads to enhancedglycogen accumulation, particularly in the liver and muscles. Akt also controls glucosehomeostasis by hampering gluconeogenesis through phosphorylation and inhibition ofa transcription factor FoxO1 [84,85]. mTORC2 indirectly acts as negative feedback of IRSthrough phosphorylating and stabilizing the ubiquitin ligase protein Fbw8, a member ofthe F-box protein family responsible for the degradation of inactive IRS1 in the cytosol, butthe mechanism needs to be further elucidated [89].

The mTOR pathway plays a key role in the regulation of cellular growth and prolifera-tion. The major cause for diet-induced obesity and insulin resistance is the overactivation ofmTOR that down-regulates insulin signaling via negative feedback regulation through thephosphorylation of IRS1 at serine residues [81,86]. Thus, pharmacological intervention thatcan target the mTOR pathway to deactivate the negative feedback loop to reverse insulinresistance, maintain nutrient homeostasis, regulate cellular growth and proliferation willbe useful in the prevention and/or treatment of diabetes. Future work should focus onenabling molecular insights related to mTOR function and regulation to develop clinicallybeneficial rational targeting of mTOR signaling.

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3.7. AMP-Activated Protein Kinase

The AMP-activated protein kinase (AMPK) is a ubiquitously expressed serine/threonine kinase. AMPK is a central regulator of multiple metabolic pathways and isactivated by diminished cellular energy state. AMPK has a heterotrimeric structure consist-ing of one catalytic subunit (α) and two regulatory subunits (β and

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Fbw8, a member of the F-box protein family responsible for the degradation of inactive IRS1 in the cytosol, but the mechanism needs to be further elucidated [89].

The mTOR pathway plays a key role in the regulation of cellular growth and proliferation. The major cause for diet-induced obesity and insulin resistance is the overactivation of mTOR that down-regulates insulin signaling via negative feedback regulation through the phosphorylation of IRS1 at serine residues [81,86]. Thus, pharmacological intervention that can target the mTOR pathway to deactivate the negative feedback loop to reverse insulin resistance, maintain nutrient homeostasis, regulate cellular growth and proliferation will be useful in the prevention and/or treatment of diabetes. Future work should focus on enabling molecular insights related to mTOR function and regulation to develop clinically beneficial rational targeting of mTOR signaling.

3.7. AMP-Activated Protein Kinase The AMP-activated protein kinase (AMPK) is a ubiquitously expressed

serine/threonine kinase. AMPK is a central regulator of multiple metabolic pathways and is activated by diminished cellular energy state. AMPK has a heterotrimeric structure consisting of one catalytic subunit (α) and two regulatory subunits (β and ϒ). Each subunit of AMPK has different isoforms encoded by distinct genes, which are expressed specifically in tissue [90,91]. Activation of AMPK restores cellular energy homeostasis by invigorating catalytic processes to generate ATP while inhibiting anabolic processes [92]. Through phosphorylation of previous metabolic substrates and transcriptional regulators, AMPK plays a substantial role in glucose uptake by upregulating GLUT4 expression through phosphorylation of transcription repressor histone deacetylase (HDAC)5, mitochondrial biogenesis, and fatty acid oxidation while suppressing the synthesis of fatty acids, cholesterol, and protein [77,93,94].

AMPK is generally correlated with the increase of insulin sensitivity in the body and decrease of insulin resistance as it is the inhibitor of acute proinflammatory responses through reduced FAO. There is a close link between dysregulation of AMPK and insulin resistance in both rodents and humans [91]. AMPK mediates contraction provoked GLUT4 translocation and glucose uptake through an insulin-independent mechanism. The cellular energy sensor Sirtuin1 and AMPK regulate each other. AMPK precedes Sirtuin 1, and they are both components of adaptive responses to insulin resistance, oxidative stress, ER stresses, and inflammation [95] (Figure 1). AMPK can be activated by exercise and calorie restriction, and in contrast, it is downregulated in response to high glucose exposure. AGE induces glucose uptake impairment in skeletal muscle through a RAGE-mediated AMPK downregulation [96]. There is a positive synergism between AGE receptor 1 (AGER1), Sirtuin 1, and AMPK. Their combined effect is responsible for blocking AGE, sustaining the expression of each other, and providing a shield against the deleterious effects of redox imbalance. Thus, AMPK and Sirtuin 1 activation is associated with a wide array of beneficial effects for maintaining glucose homeostasis in an insulin-resistant state [59]. Multiple studies have demonstrated that insulin resistance and metabolic syndrome are associated with a decrease in AMPK activity, specifically in muscles and adipose tissues [97–99].

AMPK acts as a master metabolic regulator as it is required to restore energy balance, promote glucose uptake, and guard mitochondrial health [90,100]. Moreover, AMPK also inhibits proinflammatory responses and protects against obesity-induced insulin resistance [90,101]. These multifaceted physiological effects of AMPK make it an attractive target to ameliorate pathologies related to insulin resistance. Further insights into mechanisms of AMPK regulation, post-translational modifications, and the identification of additional AMPK substrates may lead to the development of more controlled pharmacological modulations.

). Each subunit ofAMPK has different isoforms encoded by distinct genes, which are expressed specificallyin tissue [90,91]. Activation of AMPK restores cellular energy homeostasis by invigoratingcatalytic processes to generate ATP while inhibiting anabolic processes [92]. Through phos-phorylation of previous metabolic substrates and transcriptional regulators, AMPK plays asubstantial role in glucose uptake by upregulating GLUT4 expression through phosphory-lation of transcription repressor histone deacetylase (HDAC)5, mitochondrial biogenesis,and fatty acid oxidation while su7ppressing the synthesis of fatty acids, cholesterol, andprotein [77,93,94].

AMPK is generally correlated with the increase of insulin sensitivity in the body anddecrease of insulin resistance as it is the inhibitor of acute proinflammatory responsesthrough reduced FAO. There is a close link between dysregulation of AMPK and insulinresistance in both rodents and humans [91]. AMPK mediates contraction provoked GLUT4translocation and glucose uptake through an insulin-independent mechanism. The cellularenergy sensor Sirtuin1 and AMPK regulate each other. AMPK precedes Sirtuin 1, andthey are both components of adaptive responses to insulin resistance, oxidative stress, ERstresses, and inflammation [95] (Figure 1). AMPK can be activated by exercise and calorierestriction, and in contrast, it is downregulated in response to high glucose exposure. AGEinduces glucose uptake impairment in skeletal muscle through a RAGE-mediated AMPKdownregulation [96]. There is a positive synergism between AGE receptor 1 (AGER1),Sirtuin 1, and AMPK. Their combined effect is responsible for blocking AGE, sustainingthe expression of each other, and providing a shield against the deleterious effects of redoximbalance. Thus, AMPK and Sirtuin 1 activation is associated with a wide array of benefi-cial effects for maintaining glucose homeostasis in an insulin-resistant state [59]. Multiplestudies have demonstrated that insulin resistance and metabolic syndrome are associatedwith a decrease in AMPK activity, specifically in muscles and adipose tissues [97–99].

AMPK acts as a master metabolic regulator as it is required to restore energy balance,promote glucose uptake, and guard mitochondrial health [90,100]. Moreover, AMPK alsoinhibits proinflammatory responses and protects against obesity-induced insulin resis-tance [90,101]. These multifaceted physiological effects of AMPK make it an attractivetarget to ameliorate pathologies related to insulin resistance. Further insights into mech-anisms of AMPK regulation, post-translational modifications, and the identification ofadditional AMPK substrates may lead to the development of more controlled pharmaco-logical modulations.

3.8. AGE/RAGE/NF-κB Axis

Hyperglycemia is the characteristic feature of insulin resistance and one of the ma-jor factors contributing to diabetic complications, but apart from that, hyperglycemia isalso involved in a series of chemical processes known as the Maillard reaction. In theMaillard reaction, reducing sugars nonenzymatically react with the amino group of pro-teins, lipids, the nucleic acid to produce AGE [102,103]. The complexity and diversity ofAGE permanently modify the structure of proteins, plasma lipoproteins, cell membranephospholipids, or DNA. AGE can induce the formation of aggregated proteins, whichis the reason for the toxicity of AGE-modified polypeptides [104]. Mostly AGE produc-tion is endogenous, but primarily prooxidative dietary AGE can also supplement therisk for insulin resistance. AGE-mediated damage occurs through interaction with themulti-ligand, well-characterized cell surface pattern recognition receptor RAGE, whichis ubiquitously expressed on several cell types [96]. RAGE is a significant mediator inthe development of pancreatic β-cell dysfunction and the prognosis of diabetes. In thepancreas, AGE plays a mechanistic role in the aggregation of proteins to generate amyloid

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formation. Circulating AGE increases the expression of RAGE in the pancreas, which byengagement with toxic aggregates of different amyloidogenic derived proteins contributesto islet amyloidosis [8,37,105]. The key feature of islet amyloidosis is the pathologicalaggregation of IAPP, an endocrine hormone. The interaction of RAGE and IAPP activatesthe mediators of oxidative stress, inflammation, and apoptosis, which are the key featuresof IAPP induced β-cell cytotoxicity [37].

Chronic activation of RAGE by its ligands contributes to the upregulation and ac-tivation of NF-κB, a sequence-specific major transcription factor. NF-κB belongs to Relfamily proteins and plays an important role in inflammation associated with insulinresistance [106]. On activation, NF-κB increases the expression of RAGE itself and proin-flammatory cytokines, such as TNF-α, IL-1β, and IL-6, which trigger ER stress. Oncetriggered, the ER stress evokes the UPR to restore the cellular homeostasis, which increasesintracellular ROS formation [107]. Inflammation, oxidative stress, and PKC activationlead to inhibition of IRS1 activity through its enhanced serine/threonine phosphorylationand reduced tyrosine phosphorylation, which results in impaired insulin signaling, asshown in Figure 1 [108,109]. NF-κB, specifically the NF-κB p65 subunit and NF-κB p105subunit, bind to the promoter region of solute carrier family 2 member 4 (Slc2a4) gene,thus downregulating the transcription of GLUT4 expression [110,111].

The data herein show strong interdependency of hyperglycemia, insulin resistanceand RAGE/ NF-κB mediated enhancement of inflammation and oxidative stress. There is avicious cycle between inflammation and insulin resistance placing the AGE/RAGE/NF-κBaxis at the nexus of this metabolic disorder. For future research development, the majorchallenge is the design of potential therapeutic approaches that mitigate NF-κB mediatedpathological responses. This cannot be achieved without a complete understanding ofNF-κB gene regulation, and the regulatory mechanisms involved in NF-κB activation tobetter understand the long-term effects of NF-κB modulating therapies.

4. Lipotoxicity, Inflammation, and Oxidative Stress

The correlation between lipotoxicity and insulin resistance is well established andinvolves multiple pathways [16]. Lipotoxicity is the consequence of excess calorie intake,fat accumulation, and increased lipolysis, which disrupts cellular homeostasis and causesmetabolic pathway impairment in peripheral organs such as muscles, liver, pancreas, andadipose tissue [112]. The lipid spill-over of FFAs due to saturation of adipose tissue storagecapacity leads to the accumulation of fatty acyl-coenzyme A, diacylglyceride (DAG), andceramide in muscles [113]. Furthermore, prior studies have shown that these deleteriouslipid intermediates have the greatest impact on the development of skeletal muscle insulinresistance, which is responsible for 70–80% of whole-body insulin-stimulated glucoseuptake [114]. Among them, ceramide is a key lipotoxic player implicated as an antagonistof insulin action [115]. It is comprised of a sphingoid base coupled to a variable fattyacid side chain. The 4,5-trans-double bond in its sphingoid backbone bestows the uniquebiophysical properties that initiate the cellular stress responses, pro-apoptotic functions,cell growth arrest, and acts as a major inflammatory response messenger involved inthe installation of muscle insulin resistance [116,117]. This enhanced level of ceramideinterferes with muscle insulin sensitivity primarily through inhibition of the Akt/PKBactivity [113,118]. Various studies reveal the link of high plasma ceramide levels withobese insulin-resistant states [119,120]. Both in vitro and in vivo studies evaluating theintracellular concentrations of ceramides showed a positive association between ceramidein lipotoxic situations and development of insulin resistance [116,121,122]. Studies eval-uating tissue levels of ceramides by using liver [123], adipose tissues [124], and skeletalmuscle [125] biopsies also reveal that ceramide level, hyperglycemia, and insulin resistanceare positively correlated. There is a strong overlap between metabolic overload and inflam-matory signaling pathways. The group of cytokines secreted by adipose tissues is knownas adipokines [126]. These adipokines act as proinflammatory cytokines (such as IL-1,IL-6, and TNFα) that induce inflammation in the target tissue, which causes chemotactic

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invasion and infiltration of immune cells and further procreates the problem. The pro-longed abnormal secretion of cytokines will ultimately disturb the delicate balance betweeninflammation and metabolic pathways [9,127]. One of the hallmarks of prolonged lipo-toxicity is chronic low-grade metabolic inflammation, termed “metainflammation” [128].The recognition of saturated fatty acids by the Toll-Like Receptor (TLR) family of patternrecognition receptors, specifically TLR4, is another mechanism associated with obesityinduced metainflammation [129,130]. TLR4, expressed in insulin target tissues, plays acrucial role in the development of insulin resistance and inflammation. TLR4 triggersmetabolic inflammation and insulin resistance during obesity by upregulating the tran-scription of proinflammatory genes and activating proinflammatory kinases JNK, IKK, andp38. These kinases, through inhibitory phosphorylation of insulin receptor substrate (IRS)on serine residues, impair insulin signal transduction [131]. A previous study revealedthat TLR4 lies upstream of ceramide biosynthesis, which links lipid-induced inflammatorypathways to the antagonism of insulin action [132].

The activation of JNK and IKK, which induces insulin resistance through PKC, is theconsequence of activated inflammatory signaling networks [127]. There are convincingdata to support that JNK has a central role in inducing insulin resistance and type 2diabetes [133]. JNK1 knockout mice exposed to a high-fat diet confers long-term metabolicprotection from diet-induced obesity and display markedly improved insulin sensitivitywith a normal life span [134]. Direct involvement of JNK in insulin signaling is throughthe JNK phosphorylation site on IRS1 at Ser307, which inhibits the interaction of IRS1 withthe insulin receptor and constrains its tyrosine phosphorylation [133]. Furthermore, theinteraction of PKC, JNK, and IKK will disrupt the metabolic homeostasis through theactivation of kinases involved in serine phosphorylation of IRS and will inhibit insulinaction [127]. These kinases will also activate the NF-κB signaling, which will result in aburst of inflammatory responses [127,135,136].

These immune responses are also linked to another key factor crucial for the integra-tion of insulin resistance, which is known as ER stress. ER stress is accompanied by theaccumulation of misfolded protein, which activates unfolded protein response (UPR) inthe ER lumen [137]. The prolonged incapability to re-establish ER homeostasis results inthe UPR-dependent activation of inflammation and eventually, apoptosis. Mitochondriaalso become hyperactive under the nutrition overload and produce more of their naturalby-product, the reactive oxygen species. The redox imbalance occurs when the productionof reactive oxygen molecules exceeds the capacity for their clearance [18,138]. Oxidativestress is the outcome of the overgeneration of ROS in mitochondria that induce ER stress.The oxidative stress generated by ROS production, in turn, produces more oxidative stress,thus resulting in a vicious cycle of spiraling oxidative stress condition, which results in ROSinduced cellular components damage and trigger transcriptional changes that promoteinsulin resistance [139].

The excess production of reactive oxygen species directly stimulates the IKK/NF-κB,JNK pathways and damages the infrastructure of the cell through mitochondria-inducedstress responses [140]. The impact of mitochondrial perturbations will lead to downregu-lation of the key regulator of mitochondrial biogenesis peroxisome proliferator-activatedreceptor-gamma coactivator 1α (PGC-1α) and will increase mitochondrial apoptosis suscep-tibility [16,141]. The chronic elevation of FFAs is linked to inflammation through activationof protein kinase C, IKK/NF-κB, JNK pathways, ER stress, redox imbalance and resultsin dysregulation in intracellular signaling, which ultimately leads to the pathological situ-ation of insulin resistance [10,127]. The data presented herein provide directionality forthe events involved in lipotoxicity, inflammation, and oxidative stress leading to insulinresistance. During the insulin-resistant state, there are a plethora of interconnected eventstaking place at a time. Therefore, it is necessary to target several pathways at the same timefor the management of this condition. The maintenance of cellular homeostasis, mitochon-drial quality, redox homeostasis, improving the inflammatory condition, and depletion ofTLR4 protein expression might be attractive pharmacological targets to increase insulin

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sensitivity. Our understanding of mechanisms involved in insulin resistance suggeststhe use of antioxidants and pharmacological modulators suppress the chronic activationof pathways involved in the crossroad of lipotoxicity, inflammation and oxidative stress,leading to insulin resistance. Future studies delineating regulatory pathways controllingthe onset of insulin resistance will aid in identifying new target genes and facilitate thedesign of novel therapeutic approaches. The understanding of mechanistic interactionsbetween various kinases remains incomplete and needs to be evaluated further.

5. Concluding Remarks

Insulin resistance and hyperglycemia are the key pathophysiological processes of type2 diabetes mellitus and diabetic complications [11]. The principal mechanism of insulinresistance is hyperglycemia-induced hyperproduction of AGE. The high circulatory levelsof AGE lead to the ubiquitous expression of RAGE in insulin-sensitive tissues and pan-creatic β-cells. β-cell dysfunction and insulin resistance are interdependent. Diminishedpancreatic β-cells mass and dysfunction lead to a decline in insulin secretion and insulininsensitivity. Pancreatic β-cells toxicity is associated with the binding of toxic aggregates ofAGE and IAPP to the multiligand receptor RAGE. As a consequence, the production ofinflammatory cytokines, chemokines, oxidative stress, and activation of signaling cascadesrelated to cellular toxicity results in beta cell damage and degradation [37,51,102]. Theenhanced insulin demand to overcome the insulin-resistant state of the body from thepancreas with receding β-cell count further aggravates the condition.

AGE interaction with RAGE is also associated with peripheral insulin resistancewith the induction of signaling cascades causing activation of the JNK pathway, IKKα/β,and master transcription factor NF-κB [104]. All these events are associated with chroniclow-grade inflammation, ER stress inducing UPR, excess oxidative stress, activation ofstress kinases leading to IRS serine phosphorylation, degradation, and downregulationof major glucose transporter GLUT4. The downregulation of AGER1 encoded by thedolichyl-diphosphooligosaccharide protein glycosyltransferase (DDOST) gene responsiblefor AGE degradation and AMPK repression responsible for GLUT4 expression throughactivation of transcription factor MEF2, are also critical in intensifying the insulin resistancestate [96,105].

The afore-mentioned interconnected metabolic abnormalities underlying insulin-resistance mechanism leads to the pathological condition of diabetes mellitus. Multiplepathophysiological mechanisms underlying insulin resistance should be targeted andtreated simultaneously to combat this metabolic dysfunction. This review concludes thatfuture strategic interventions focused on reducing AGE production and downregulatingRAGE expression along with targeting insulin sensitivity will be beneficial to counteractthe insulin signaling pathway perturbations. This approach will be beneficial not only fromthe perspective of improved insulin sensitivity but also from the perspective of preservingthe β-cell mass and function.

Author Contributions: M.K. and A.A. conceptualized the idea. M.K. took the lead in writing themanuscript. A.A. reviewed the content and approved the final version of this manuscript. J.A. andM.A.B.K. provided critical feedback and helped shape the manuscript. All authors have read andagreed to the published version of the manuscript.

Funding: This research received no external funding.

Acknowledgments: The authors acknowledge CUSABIO Technology LLC for using some of themarkers from their article AGE-RAGE signaling pathway in the key in Figure 1.

Conflicts of Interest: The authors declare that there are no conflicts of interest.

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